Andrographis paniculata extract

ABSTRACT

An extract of  Andrographis paniculata  extract containing androgapholide, 14-deoxyandrographolide, 14-deoxy-11,12-dehydroandrographolide, neoandrographolide, polysaccharides, and flavanoids. Also disclosed is a pharmaceutical composition containing such an extract and its use for treating inflammatory bowel disease.

BACKGROUND

Inflammatory bowel disease includes chronic gastrointestinal disorderscharacterized by infiltration of inflammatory cells into the mucosa ofthe digestive tract. Ulcerative colitis and Crohn's disease are twoprevalent conditions among them.

Ulcerative colitis takes place in the large intestine (i.e., colon). Theinner lining of the disordered intestine becomes inflamed and developsulcers.

Crohn's disease most commonly affects the end of the small intestine(i.e., terminal ileum) and parts of the large intestine. It causesinflammation that extends much deeper into the layers of the intestinalwall than ulcerative colitis.

Both ulcerative colitis and Crohn's disease are attributed todysregulation of pro-inflammatory cytokine, including TNFα and IL-1β.See, e.g., McClane S. J. et al., Journal of Parenteral and EnteralNutrition 23, 1999. Therapeutic agents have been developed based ondown-regulation of pro-inflammatory cytokine. For example,5-aminosalicylic acid, an inhibitor of TNFα signaling events, has beenused to treat ulcerative colitis. See, e.g., Therapeutic Immunology Ed.Austen, K F., Blackwell Publishing, 2001, 159-167. However, mostinflammatory bowel disease therapeutics have limited efficacy orsignificant side effects.

There is still a need to develop more effective therapeutic agents fortreating inflammatory bowel disease.

SUMMARY

This invention is based on a surprising finding that an extract ofAndrographis paniculata effectively exerts a curative effect againstinflammatory bowel disease. The extract contains andrographolidelactones, polysacchorides, and flavanoids; constituting 10-22%(preferably 13-17%), 18-28% (preferably 20-25%), and 10-15% (preferably12-14) of the dry weight of the extract, respectively. Theandrographolide lactones include andrographolide,14-deoxyandrographolide, 14-deoxy-11,12-dehydroandrogapholide, andneoandrographolide, which constitute 2-20% (preferably 3-10%, morepreferably 6-10%), 0.01-6% (preferably 0.01-2%, more preferably0.01-1%), 1-6% (preferably 2-5%, more preferably 2-4%), and 1-5%(preferably 2-4%) of the dry weight of the extract, respectively.

Another aspect of this invention relates to a method of treatinginflammatory bowel disease (including Crohn's disease and ulcerativecolitis). The method includes administering to a subject in need of thetreatment an effective amount of the above-described extract.

Also within the scope of this invention are a pharmaceutical compositioncontaining the extract described above and a pharmaceutically acceptablecarrier, the use of such a composition to treat inflammatory boweldisease, and the use of such a composition for the manufacture of amedicament for treating this disease.

Details of several embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description, and also from theclaims.

DETAILED DESCRIPTION

To prepare the extract of this invention, one can immerse the aerialpart of Andrographis paniculata in 80-95% ethanol, collect the ethanolphase, and then remove the ethanol. An actual example is provided below.The extract thus obtained can be further purified by thin layerchromatography, flash column chromatography, high. performance liquidchromatography, or any other suitable methods.

This invention includes methods of treating inflammatory bowel diseaseby administering to a subject in need thereof an effective amount of theextract of this invention. The term “an effective amount” refers to theamount of the extract which is required to confer one of theabove-described therapeutic effects in the subject. Effective amountsmay vary, as recognized by those skilled in the art, depending on routeof administration, excipient usage, and the possibility of co-usage withother agents. Preferably, the effective amount is 1-100 mg/kg/day basedon the dry weight of the extract. The term “treating” refers toadministering the extract to a subject that has inflammatory boweldisease, or has a symptom of the disease, or has a predisposition towardthe disease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the disease, the symptoms of thedisease, or the predisposition toward the disease.

To practice one of the above-described methods, one administers to asubject in need thereof orally, rectally, parenterally, by inhalationspray, or via an implanted reservoir a composition that is either theabove-mentioned extract alone or a mixture of the extract and apharmaceutically acceptable carrier. The term “parenteral” as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrastemal, intrathecal, intralesional and intracranial injection orinfusion techniques.

An oral composition can be any orally acceptable dosage form including,but not limited to, tablets, capsules, emulsions and aqueoussuspensions, dispersions and solutions. Commonly used carriers fortablets include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added to tablets. Tablets mayalso be coated for delivery or cosmetic effects. For oral administrationin a is capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally,the active ingredient can be suspended or dissolved in an oily phasecombined with emulsifying or suspending agents. If desired, certainsweetening, flavoring, or coloring agents can be added.

A rectal composition can be any rectally acceptable dosage formincluding, but not limited to, cream, gel, emulsion, suspension,suppository, and tablet. One preferred dosage form is a suppositoryhaving a shape and size designed for introduction into the rectalorifice of the human body. A suppository usually softens, melts, ordissolves at body temperature. Suppository excipients include, but arenot limited to, theobroma oil (cocoa butter), glycerinated gelatin,hydrogenated vegetable ails, mixtures of polyethylene glycols of variousmolecular weights, and fatty acid esters of polyethylene glycol.

A sterile injectable composition (e,g., aqueous or oleaginoussuspension) can be formulated according to techniques known in the artusing suitable dispersing or wetting agents (such as, for example, Tween80) and suspending agents. The sterile injectable preparation can alsohe a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol.

Among the acceptable vehicles and solvents that can be employed aremannitol, water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents.

An inhalation composition can be prepared according to techniques wellknown in the art of pharmaceutical formulation and can be prepared assolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A topical composition can be formulated in form of oil., cream, lotion,ointment and the like. Suitable carriers for the composition includevegetable or mineral oils, white petrolatum (white soft paraffin),branched chain fats or oils, animal fats and high molecular weightalcohols (greater than C12). The preferred carriers are those in whichthe active ingredient is soluble. Emulsifiers, stabilizers, humectantsand antioxidants may also be included as well as agents imparting coloror fragrance, if desired. Additionally, transdermal penetrationenhancers may be employed in these topical formulations. Examples ofsuch enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762.Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil, such as almond oil,is admixed. An example of such a cream is one which includes about 40parts water, about 20 parts beeswax, about 40 parts mineral oil andabout 1 part almond oil. Ointments may be formulated by mixing asolution of the active ingredient in a vegetable oil, such as almondoil, with warm soft paraffin and allowing the mixture to cool. Anexample of such an ointment is one which includes about 30% almond andabout 70% white soft paraffin by weight.

A carrier in a pharmaceutical composition must be “acceptable” in thesense of being compatible with the active ingredient of the formulation(and preferably, capable of stabilizing it) and not deleterious to thesubject to be treated. For example, solubilizing agents, such ascyclodextrins (which form specific, more soluble complexes with one ormore of active compounds of the extract), can be utilized aspharmaceutical excipients for delivery of the active compounds. Examplesof other carriers include colloidal silicon dioxide, magnesium stearate,cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

A suitable in vitro assay can be used to preliminarily evaluate theefficacy of the above-described extract in inhibiting expression of TNFαor IL-1β. The extract can further be examined for its efficacy intreating inflammatory bowel disease by in vivo assays. For example, theextract can be administered to an animal (e.g., a mouse model) 1_(.)0 orhuman having inflammatory bowel disease and its therapeutic effects arethen accessed. Based on the results, an appropriate dosage range andadministration route can also be determined.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificexamples are, therefore, to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever. Allof the publications, including patents, cited herein are herebyincorporated by reference in their entirety.

Example 1 Preparation of the Andrographis paniculata Extract

Dry powder of Andrographis paniculata leaves (350 kg) was immersed in90% ethanol (2,100 kg). The mixture was refluxed at 75-80° C. for twohours. The ethanol. phase was collected and the solid residue wassubjected to extraction again. The ethanol solutions were combined,filtered, and concentrated to afford a wet mixture having a density of1.00-1.10 g/ml.

A small amount of the mixture was dried and analyzed for its compositionby high performance liquid chromatography and spectrophotometry. Theresult showed that the dry extract contained andrographolide lactones(14.8% of the dry weight of the extract), polysacchrides (24.6%) andflavanoids (12.8%). Among the andrographolide lactones, andrographolidewas at 9.2% of the dry weight of the extract, 14-deoxyandrographolide at<0.1%, 14-deoxy-11,12-dehydroandrographolide 2.6%, andneoandrographolide 3.0%. Dextrin was added (0.03 kg) to the wet mixture,which was then spray-dried (inlet: 185-195° C.; outlet: 90-100° C.). Thesolid extract thus obtained was ground, sieved, and packaged to formtablets and capsules as described below.

Tablets were prepared as follows. Starch (10 g) and sugar (10 g) weremixed with purified water (80.0 g) to yield a paste. Separately, theextract (500.0 g), starch (140.0 g), microcrystalline cellulous (337.5g), and the paste were mixed, wet granulized, and dried at 55° C. Thedried granules (957.6 g) and magnesium stearate (2.4 g) were mixed for 5minutes. The final mixture was compressed to form tablets (400mg/tablet, eqv. to 200 mg the extract/tablet). The tablets werefilm-coated with a paste prepared by mixing hypromellose (7.5 g),propylene glycol (1.6 g), titanium dioxide (3.0 g), Food Drug & toCosmetic color lake (0.4 g), and purified water (87.5 g) to afford thedesired Andrographis paniculata extract-containing tablets.

Capsules were prepared as follows. The extract (340.0 g), pre-driedstarch (221.0 g), silicon dioxide (2.125 g), and microcrystallinecellulous (34.0 g) were mixed. The mixture was filled into #0 hard-shellcapsules using a capsule filling board to form is the desiredAndrographis paniculata extract-containing capsules (351.25 mg themixture/capsule, eqv. to 200 mg the extract/capsule).

Example 2 Inhibition of TNFα and IL-1β Expression

Peripheral blood monocytes (PBMC) cells are isolated from fresh bloodusing Ficoll-Paque Plus (Amersham Bioscience), according to the protocolprovided by the manufacturer. The cells are suspended in RPMI 1640 mediacontaining 10% FBS at a concentration of 1×1.0⁵ cells/ml and seeded in a96-well plate. Each reaction is carried out in three wells.

10 μl of the extract of Andrographis paniculata in DMSO is added intoeach well (final concentrations: 0.1, 0.3, 1, 3, 10, and 30 μg/ml).Dexamethason (final concentration: 10 μM) is used as positive control.10 μl of the media is used as a negative control. The plate is incubatedat 37° C. under 5% CO₂ for 15 minutes. After 10 μl aliquots of 100 μg/mllipopolysaccharide are added to all wells except for the negativecontrols, the plate is incubated at 37° C. under 5% CO₂ overnight.

The plate is spun at 1000 rpm for 15 minutes and the supernatants arecollected. Concentrations of TNFα and IL-1β are measured using the TNFαELISA (Enzyme Linked Immunosorbent Assay) Kit and IL1-β ELISA Kit(Jingmei Bioengineer Technology).

The inhibition ratio is calculated as follows:

${{Inhibition}\mspace{14mu} {Ratio}\mspace{14mu} (\%)} = {\left( {1 - \frac{C_{extract} - C_{control}}{C_{LPS} - C_{Control}}} \right) \times 100}$

where C_(extract) is the concentration of TNFα or IL-1β in PBMC cellstreated with the extract and LPS, C_(LPS) is the concentration of TNFαor IL-1β in PBMC cells treated with LPS and dexamethason, andC_(control) is the concentration of TNFα or IL-1β in PBMC cells withoutbeing treated with LPS or the extract.

Example 3 Treatment of Inflammatory Bowel Disease in Mouse Model

Balb/c male mice (18-24 g, purchased from Chinese Academy of Scienceanimal center) are anaesthetized with 1% pentobarbital sodium at 0.05mg/10 g. 1.5 mg of 2,4,6-trinitrobenzenesulfonic acid in 50% ethanol isadministered slowly to each mouse (except blank control mice) via acatheter to induce inflammatory bowel disease. Blank control mice onlyreceive 0.1 ml of 50% ethanol. The mice are treated with the test sample24 hours and 2 hours prior to the inflammatory bowel diseaseadministration and daily for 5 days after the administration.

The body weight of each mouse is monitored every day before and afterthe 2,4,6-trinitrobenzenesulfonic acid administration. The mice aresacrificed 24 hours after the last administration of test samples.Colons are removed and weighed. Furthermore, the colon weight to bodyweight ratio is calculated and adhesion between colon and other organsis also monitored.

Samples of colon tissues located precisely 2 cm above the anal canal areobtained, fixed in 10% buffered phosphate, embedded in paraffin,sectioned, and stained with hematoxylin/eosin. The degree ofinflammation on microscopic cross sections is graded from 0 to 4 (0: nosigns of inflammation; 1: a very low level of inflammation; 2: a lowlevel of leukocyte infiltration; 3: a high level of leukocyteinfiltration, a high vascular density, and a thickened colon wall; and4: transmural infiltrations, loss of goblet cells, a high vasculardensity, and a thickened colon wall).

Example 4 Clinical Treatment of Ulcerative Colitis

To study the efficacy of the Andrographis paniculata extract in treatingulcerative colitis, a randomized, double-dummy, active controlled 8-weekclinical trial was conducted at 5 locations in Shanghai, China incompliance with to the International Conference on Harmonisation-GoodClinical Practice (ICH-GCP) guidelines, 120 patients withcolonoscopy-confirmed mildly to moderately active ulcerative colitiswere assigned to two groups (60 patients/group). One group was treatedwith the Andrographis paniculata extract-containing tablets mentionedabove (3 times daily, 2 tablets each time,), and the other was treatedwith 5-amino-2-hydroxybenzoic acid, i.e., Etiasa (3 times daily, 500-mggranule each time,). All other medications were excluded. Thetherapeutic effects were assessed biweekly using a scale similar to thepartial Mayo Scoring System, and the clinical symptom score reduction(≧50% reduction in symptoms) was calculated. Scores were thenretrospectively calculated using the standard partial Mayo scores (PMS),clinical, response (improvement ≧2 points or final score of 0) andremission (≦1 PMS score at week 8). Colonoscopies at the beginning andat the end of treatment were rated with a modified Baron score, andbiopsies taken during colonoscopy were graded histologically with ascale of 0-3.

Patients in the two groups had similar demographics. In each group, themean duration of disease ranged from 3.5-3.7 years and the baseline meanPMS was 3.8. En the 53 intent-to-treat patients treated with theextract, the clinical symptom score reduction was 27% in the patients atweek 2 and improved to 56% in the patients by week 8. The 55intent-to-treat Etiasa treated patients showed similar reduction. Theclinical response rate at week 8 was 58% in the patients treated withthe extract and 58% in the patients treated with Etiasa. The remissionrate at week 8 was 43% in the patients treated with the extract and 58%in the patients treated with Etiasa. The results of PMS at the baselineand week 8 in both groups are statistically significant (p<0.0002).

Endoscopically, at week 8, 28% of the patients treated with the extractand 24% of those treated with Etiasa were in complete remission (Baronscore of 0); and 47% of the patients treated with the extract and 42% ofthose treated with Etiasa had scores reduced by at least two grades.

Histologically, 19 of the patients treated with the extract and 15 ofthe patients treated with Etiasa were evaluated. 10 of the 19 patientstreated with the extract showed decrease of inflammation scores by25-50% at week 8, and so did 6 of the 15 patients treated with Etiasa.In the extract-treated group, 12/15 entering with elevated C-reactiveprotein levels showed normal levels at week 8, compared to 4/6 in theEtiasa-treated group. The results in both groups are statisticallysignificant (p<0.0001).

The results indicated that the extract was effective in treatingulcerative colitis. Surprisingly, its efficacy was comparable to or evenbetter than Etiasa.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are also within the scope of thefollowing claims.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

1-25. (canceled)
 26. A method of effectively treating an inflammatorybowel disease in a human comprising orally administering the human inneed thereof, in a solid dosage form chosen from tablets and capsules,said solid dosage form comprising an extract of Andrographis paniculatacomprising andrographolide lactones, wherein the andrographolidelactones in said extract comprise: andrographolide in an amount rangingfrom 2% to 20% of the dry weight of the extract; 14-deoxyandrographolidein an amount ranging from greater than 0 to 2% of the dry weight of theextract; 14-deoxy-11,12-dehydrogen-andrographolide in an amount rangingfrom 1% to 6% of the dry weight of the extract; and neoandrographolidein an amount ranging from 1% to 5% of the dry weight of the extract,further wherein said solid dosage form further comprises dextrin and atleast one pharmaceutically acceptable carrier, and even further whereinthe method of effectively treating an inflammatory bowel disease in ahuman is conducted for 8 weeks by orally administering a daily dosage ofat least 1200 mg to said human.
 27. The method of claim 26, wherein saidextract further comprises polysaccharides and flavonoids.
 28. A methodof effectively treating ulcerative colitis in a human comprising orallyadministering the human in need thereof, in a solid dosage form chosenfrom tablets and capsules, said solid dosage form comprising an extractof Andrographis paniculata comprising andrographolide lactones, whereinthe andrographolide lactones in said extract comprise: andrographolidein an amount ranging from 2% to 20% of the dry weight of the extract;14-deoxyandrographolide in an amount ranging from 0.01% to 2% of the dryweight of the extract; 14-deoxy-11,12-dehydrogen-andrographolide in anamount ranging from 1% to 6% of the dry weight of the extract; andneoandrographolide in an amount ranging from 1% to 5% of the dry weightof the extract, further wherein said solid dosage form further comprisesdextrin and at least one pharmaceutically acceptable carrier, and evenfurther wherein the method of effectively treating ulcerative colitis ina human is conducted for 8 weeks by orally administering a daily dosageof said solid dosage form, and even further wherein said daily dosage,following said oral administration to said human, has been shown to beeffective for treating ulcerative colitis in a human.
 29. The method ofclaim 28, wherein said extract further comprises polysaccharides andflavonoids.
 30. A method of effectively treating ulcerative colitis in ahuman comprising orally administering the human in need thereof, in asolid dosage form chosen from tablets and capsules, said solid dosageform comprising an extract of Andrographis paniculata comprisingandrographolide lactones, wherein the andrographolide lactones in saidextract comprise: andrographolide in an amount ranging from 2% to 20% ofthe dry weight of the extract; 14-deoxyandrographolide in an amountranging from 0.01% to 2% of the dry weight of the extract;14-deoxy-11,12-dehydrogen-andrographolide in an amount ranging from 1%to 6% of the dry weight of the extract; and neoandrographolide in anamount ranging from 1% to 5% of the dry weight of the extract, furtherwherein said solid dosage form further comprises at least onepharmaceutically acceptable carrier, and even further wherein the methodof effectively treating ulcerative colitis in a human is conducted for 8weeks by orally administering to said human a dosage effective fortreating said ulcerative colitis in said human, and even further whereinsaid solid dosage form, following said oral administration to saidhuman, shows efficacy in treating said ulcerative colitis.
 31. Themethod of claim 30, wherein said extract further comprisespolysaccharides and flavonoids.
 32. A method of effectively treatinginflammatory bowel disease in a human comprising orally administeringthe human in need thereof, in a solid dosage form chosen from tabletsand capsules, said solid dosage form comprising an extract ofAndrographis paniculata comprising andrographolide lactones, wherein theandrographolide lactones in said extract comprise: andrographolide in anamount ranging from 2% to 20% of the dry weight of the extract;14-deoxyandrographolide in an amount ranging from 0.01% to 2% of the dryweight of the extract; 14-deoxy-11,12-dehydrogen-andrographolide in anamount ranging from 1% to 6% of the dry weight of the extract; andneoandrographolide in an amount ranging from 1% to 5% of the dry weightof the extract, further wherein said solid dosage form further comprisesat least one pharmaceutically acceptable carrier, and even furtherwherein the method of effectively treating inflammatory bowel disease ina human is conducted for 8 weeks by orally administering to said human adosage effective for treating said inflammatory bowel disease in saidhuman.
 33. The method of claim 32, wherein said extract furthercomprises polysaccharides and flavonoids.